Although it is known that individual cells (blastomeres) of a developing mammalian embryo prior to the blastocyst implant stage are capable of developing into totipotent embryos, manipulation of embryos in the 2-, 4-, or 8-cell morulae stage has met with limited experimental success and has not developed into a commercial practice. Moore, et al. (1968) reported on a series of experiments with rabbit ova in which individual blastomeres were cultured in their own ova membrane (the zona pellucida). The culturing was compared with that of nude blastomeres without zona, and separated blastomeres inserted in the host zona. The culturing of the blastomeres was carried out in the fallopian tubes of the recipient does. No single blastomere devoid of zona survived, but survival was obtained with some of the blastomeres separated from the 2-, 4-, and 8-cell ova enclosed in their own zona, and the surviving blastomeres developed into normal rabbits. Separation of the blastomeres at the 2-cell stage gave a higher rate of survival (30%) than those separated at the 4- and 8-cell eggs (19% and 11%). Single blastomeres of 4-cell ova inserted in host zona showed limited development, undergoing one or more cleavages.
Isolated blastomeres and blastomere clusters prepared by microsurgery from pre-implantation embryos require some form of protection when the zona pellucida is removed or its integrity compromised (Moore, et al., 1968; Willadsen, 1979). Agar has been used to seal or encapsulate ruptured zona pellucida containing blastomeres (Willadsen, 1979). In the experiments reported by Willadsen, 2-celled sheep embryo were manipulated by severing the zona pellucida, removing and separating the blastomeres, and re-inserting a single blastomere into an evacuated zona. The zona-containing blastomeres were transferred to an agar solution held in the tip of a pipette until the solution hardened. A tiny solid cylinder of the agar containing the zona-encased blastomere was ejected. The small cylinders were encapsulated in larger solid cylinders of agar. The cylinders were transferred to ewe oviducts for incubation. After reaching the late morulae or early blastocyst stage, the agar cylinders were recovered, the agar was removed, and the embryos were implanted. Some live offspring were produced. The reduced embryo survival rate compared to standard embryo transfer was attributed to the difficulty of manipulating the embryos in the agar, which required the use of hypodermic needles, and other procedures.
The embedding of embryos in agar is referred to in the literature as the agar "chip" technique. The micromanipulations involved in the agar chip technique were summarized by Willadsen in 1982 in a treatise on "Mammalian Egg Transfer". After referring to the disappointing results in experiments on the developmental potential of isolated blastmeres, Willadsen concluded "with near certainty that the central problem in all instances arose from the lack of adequate methods for culture of the micromanipulated embryos in vitro in line with the inability of the embryonic cells to survive in vivo after the zona pellucida had been ruptured or removed". He further concluded that "none of the methods currently used is entirely satisfactory for the culture of early embryos, i.e., cleavage stages which are most dependent on the zona pellucida for their survival in vivo."
With reference to agar embedding, Willadsen (1982) had reported that "single blastomeres may be embedded without a zona pellucida, but this is not advisable, because it makes it very difficult to release the embryos at a later stage". Willadsen recommended that isolated blastomeres be contained in their own zona or in host zona, and that agar chip embedding be used only for such zona-contained blastomeres.
Egestone et al. (1985) reported on experiments in which 1-cell bovine embryos were embedded in agar and successfully cultured to the blastocyst stage in ewe oviducts. A tendency of the chips to dissolve in the oviduct fluid was observed. Boland (1984) reported on experiments using the rabbit oviduct as a screening tool for the viability of mammalian eggs embedded in agar chips. He concluded that the rabbit oviducts were unsuitable for such screening "because of the high rate of degradation of agar chips". More recently, Adaniya, et al. (1987) reported on the coating of rabbit embryos ready for implantation with sodium alginate. Following implantation in the uterus, the rate of degradation of the sodium alginate was observed. It was found that after four hours only 38% of the capsules remained, and after six hours none of the capsules were recoverable.